Troubleshooting Guide to Expressing Intrinsically Disordered Proteins for Use in NMR Experiments

Intrinsically disordered proteins (IDPs) represent a structural class of proteins that do not have a well-defined, 3D fold in solution, and often have little secondary structure. To characterize their function and molecular mechanism, it is helpful to examine their structure using nuclear magnetic resonance (NMR), which can report on properties, such as residual structure (at both the secondary and tertiary levels), ligand binding affinity, and the effect of ligand binding on IDP structure, all on a per residue basis. This brief review reports on the common problems and decisions that are involved when preparing a disordered protein for NMR studies. The paper covers gene design, expression host choice, protein purification, and the initial NMR experiments that are performed. While many of these steps are essentially identical to those for ordered proteins, a few key differences are highlighted, including the extreme sensitivity of IDPs to proteolytic cleavage, the ability to use denaturing conditions without having to refold the protein, the optimal chromatographic system choice, and the challenges of quantifying an IDP. After successful purification, characterization by NMR can be done using the standard 15N-heteronuclear single quantum coherence (15N-HSQC) experiment, or the newer CON series of experiments that are superior for disordered proteins

Investigation of a transcription factor complex and intrinsically disordered proteins

LIM domain binding protein 1 (Ldb1) is a chromatin looping factor that forms part of a transcriptional ‘pentameric complex’. Ldb1 contains two domains that are essential for looping: a self-association domain, and a LIM interaction domain (LID) that binds LIM proteins such as Lmo2, which in turn binds to DNA binding transcription factors, Tal1, E2a and Gata-1. It was proposed that the Gata-1 binding protein FOG1 could bind to the pentameric complex. GFP-tagged FOG1 was shown to bind the complex by multi angle laser light scattering, providing a mechanism by which the intrinsically weak Gata-1/FOG1 interaction is bolstered through binding to other units of the pentameric complex. Little is known about proteins that are distantly related to mammalian Ldb1. Two such proteins, Ldb1 from C. elegans and Adn1 from S. pombe were expressed in bacteria. They had generally poor solubility, but use of a maltose binding protein tag promoted solubility, and different expression systems may enable their further study. Ldb1 was reported to bind the E3 ubiquitin ligase protein, RLIM. No interaction could be detected between these proteins by yeast two-hybrid analysis using truncated or full length proteins. The interaction was detected in mammalian cells using FLAG pull-down experiments, but truncation mutants of these proteins could not be expressed. RLIM has high levels of predicted disorder, which may contribute to its degradation in both cell types. An assay was developed in which dimerization domains could stabilise disordered binding regions. Constructs containing GST, a coiled-coil domain of CtIP, or the leucine zipper of GCN4, were tethered to a test peptide and assayed for binding in yeast two-hybrid assays. The domains from CtIP and/or GCN4 allowed the interaction to be detected. Although the assay could not detect an interaction between RLIM and Ldb1, it shows promise for detecting interactions for disordered proteins, and can be adapted to different expression systems

Investigation of a transcription factor complex and intrinsically disordered proteins

LIM domain binding protein 1 (Ldb1) is a chromatin looping factor that forms part of a transcriptional ‘pentameric complex’. Ldb1 contains two domains that are essential for looping: a self-association domain, and a LIM interaction domain (LID) that binds LIM proteins such as Lmo2, which in turn binds to DNA binding transcription factors, Tal1, E2a and Gata-1. It was proposed that the Gata-1 binding protein FOG1 could bind to the pentameric complex. GFP-tagged FOG1 was shown to bind the complex by multi angle laser light scattering, providing a mechanism by which the intrinsically weak Gata-1/FOG1 interaction is bolstered through binding to other units of the pentameric complex. Little is known about proteins that are distantly related to mammalian Ldb1. Two such proteins, Ldb1 from C. elegans and Adn1 from S. pombe were expressed in bacteria. They had generally poor solubility, but use of a maltose binding protein tag promoted solubility, and different expression systems may enable their further study. Ldb1 was reported to bind the E3 ubiquitin ligase protein, RLIM. No interaction could be detected between these proteins by yeast two-hybrid analysis using truncated or full length proteins. The interaction was detected in mammalian cells using FLAG pull-down experiments, but truncation mutants of these proteins could not be expressed. RLIM has high levels of predicted disorder, which may contribute to its degradation in both cell types. An assay was developed in which dimerization domains could stabilise disordered binding regions. Constructs containing GST, a coiled-coil domain of CtIP, or the leucine zipper of GCN4, were tethered to a test peptide and assayed for binding in yeast two-hybrid assays. The domains from CtIP and/or GCN4 allowed the interaction to be detected. Although the assay could not detect an interaction between RLIM and Ldb1, it shows promise for detecting interactions for disordered proteins, and can be adapted to different expression systems

Paramagnetic chemical probes for studying biological macromolecules

Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.Macromolecular Biochemistr

Dynamique fonctionnelle des protéines: études d'une lipase et d'une protéine A de la membrane externe de bactérie.

Understanding the function of proteins and biological systems requires an accurate knowledge of the underlying molecular mechanisms. Crystallography and nuclear magnetic resonance provide a detailed description of these mechanisms, with an atomic resolution, by providing data on both structures and motions. We investigated two proteins, the lip2 lipase from the yeast Yarrowia lipolytica and the membrane protein OmpA from the bacteria Klebsiella pneumoniae. We tried to produce lip2 with uniform and amino-acid specific stable isotope labelling on its functional loop (the lid) for NMR experiments. The homologous recombinant expression in Yarrowia lipolytica turned out to be the most efficient for uniform labelling but failed for specific labelling due to extensive isotope scrambling. We solved the structure of OmpA C-terminal domain by X-ray crystallography, and analyzed its dynamics in solution by NMR (15N relaxation techniques). We characterized its transmembrane N-terminal domain in proteoliposomes by solid state NMR: using state of the art ultra-fast MAS (60 kHz), 1H detection and a 1 GHz spectrometer, we could assign most β-barrel resonances and establish a NH order parameter profile. In a complementary approach, we used proteolysis to reveal a unique trypsin cleavage site on the extracellular loop 3. Finally, a first characterization of the full-length protein expressed in the outer membrane of Escherichia coli was initiated by solid state NMR on intact outer membranes.La compréhension de la fonction des protéines et des systèmes biologiques passe par une connaissance fine des mécanismes moléculaires sous-jacents. La cristallographie et la résonance magnétique nucléaire permettent d’appréhender ces mécanismes au niveau atomique en fournissant des informations sur la structure et sur la dynamique des macromolécules biologiques. Nous nous sommes ainsi intéressés à deux protéines, la lipase lip2 de la levure Yarrowia lipolytica et la protéine membranaire OmpA de la bactérie Klebsiella pneumoniae. Nous avons recherché des conditions d’expression de la protéine lip2 marquée uniformément ou spécifiquement sur une boucle (appelée « lid ») afin d’en étudier la dynamique. Des conditions de marquage uniforme à l’azote 15 de lip2 recombinante dans Yarrowia lipolytica ont été mises au point, mais le marquage acide aminé spécifique n’a pu être réalisé à cause de phénomènes de dilution isotopique trop importants dans cette levure. Nous avons résolu par cristallographie aux rayons X la structure du domaine C-terminal de la protéine OmpA et étudié sa dynamique en solution par RMN (techniques de relaxation 15N). Nous avons caractérisé la dynamique de son domaine N-terminal membranaire reconstitué en liposomes par RMN du solide : en utilisant la rotation à l’angle magique à 60kHz et à la détection 1H sur un spectromètre 1 GHz, nous avons pu attribuer une majorité des résonances du tonneau β et établir un profil de paramètre d’ordre des vecteurs NH. Des expériences de protéolyse ménagée ont révélé par ailleurs un site de coupure unique à la trypsine au sein de la boucle extracellulaire L3. Enfin, une première caractérisation de la protéine complète exprimée dans la membrane externe d’Escherichia coli a été entreprise par RMN du solide sur membranes externes natives